Method for decreasing immunogenicity

ABSTRACT

A method for decreasing the immunogenicity of antibody variable domains is disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/711,181, filed Sep. 21, 2017 (now allowed), which is a divisional ofU.S. patent application Ser. No. 14/308,936, filed Jun. 19, 2014 (nowU.S. Pat. No. 9,803,027), which is a divisional of U.S. patentapplication Ser. No. 12/973,968, filed Dec. 21, 2010 (now U.S. Pat. No.8,796,425), which claims priority to U.S. Provisional Patent ApplicationSer. No. 61/289,446 filed Dec. 23, 2009, the entire contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a method of altering the immunogenicity ofantibody varies able domains, in particular of scFvs.

BACKGROUND ART

Therapeutic antibodies administered to a subject in need are oftenrecognized as foreign by the subject's immune system. Even if theadministered antibodies have been humanized, e.g. by grafting of murineCDRs into human immunoglobulin frameworks to minimize the mousecomponent, they still may elicit an immune response which compromisesthe efficacy and/or safety of the therapeutic.

According to the literature antibody responses in patients are dependenton the presence of both B-cell epitopes and T-cell epitopes. When aB-cell receptor recognizes and binds an antigen such as an administeredtherapeutic antibody, the antigen is internalized into the B cell byreceptor-mediated endocytosis and undergoes proteolytic processing. Theresulting peptides are subsequently presented by MHC class II molecules.Upon recognition of the T cell epitope by a T helper cell, the latterstimulates the corresponding B cells to proliferate and differentiateinto antibody producing plasma cells.

In order to decrease the response of the patient's immune system to theadministered antibodies, the prior art has provided severalde-immunization techniques. Most of the current approaches focus on theremoval of T-cell epitopes, whereas there are only limited examples ofmethods to reduce B-cell immunogenicity.

WO 93/18792 describes a process for the modification of antibodies bypartial reduction of the antibody. This alters their immunogenicity sothat their ability to induce an anti-isotypic response is selectivelydiminished, while they remain able to elicit an anti-idiotypic response.Albeit the method would be suitable for vaccines, anti-idiotypicresponses are not desirable for other therapeutic applications.

Molineux G (2003) Pharmacotherapy 23: 35-85 describes the coupling ofproteins to high-molecular-weight polyethylene glycol. However, Onda, M.et al (2008), PNAS Vol 105(32): 11311-11316 have reported a limitedsuccess of this approach with hybrid proteins composed of the variablefragment attached to a bacterial or plant toxin. Their hybrid proteinswere inactivated; moreover, they found only a minor decrease inimmunogenicity.

A second approach consists in chemotherapy prior to antibodyadministration, wherein patients are treated with cyclophosphamide orfludarabine. This approach is not desirable for the patients as thetreatment damages the immune system (Kusher, B H et al (2007), PediatrBlood Cancer 48: 430-434; Leonard J P et al (2005), J Clin Oncol 23:5696-5704).

Nataga, S. and Pastan, I. (2009), Adv Drug Deliv Rev, p. 977-985 andOnda, M. et al (2008), PNAS Vol 105(32): 11311-11316 propose pointmutations at “antigenic hot spots” on the foreign protein surface,thereby removing the B-cell epitope. They substituted bulky hydrophilicresidues with large exposed areas by small amino acids (alanine, glycineand serine). Alanine is preferred for substitution as it is typicallypresent in buried and exposed positions of all secondary structures andalso does not impose new hydrogen bonding. Alanine lacks side chainatoms after the β-carbon that can react with antibodies and moreovermaintains the conformation of the antigen. However, said “hot spots”described by Nataga and Pastan are conformational epitopes which arelocated in discrete clusters on the protein surface. Extensiveexperimental work is needed to determine the locations of the epitopesthat could not be reproduced in a computer simulation and thus, theirmethod does not represent a general solution to reduce immunogenicity ofantibodies that can be applied routinely. Furthermore, a principleassumption of this method is that mainly hydrophilic residues on themolecular surface are involved in the contact with the host antibody.For most foreign proteins this is in fact true, however in cases wereonly portions (e.g. fragments, domains) of a naturally occurring proteinis used, it may well be that also hydrophobic amino acids, formerlyshielded by the contact to other domains become exposed to the solventand present as epitope to the immune system. This is explicitly the casefor Fv antibody fragments, where the interface residues on the variabledomain are covered in the Fab fragment but are exposed in isolatedvariable domains. Currently available algorithms to predict B cellepitopes are poorly validated and typically have a low rate of success.

Thus, there is a need in the art to provide straight forward methodswhich effectively reduce the immunogenicity of antibody fragments andparticularly for the variable domains.

SUMMARY OF THE INVENTION

Hence, it is a general object of the invention to provide a method todecrease the immunogenicity of any antibody variable domain without theneed to perform extensive molecular modeling efforts. In particular, itis an object of the invention to provide a method to remove B-cellepitopes from antibody variable domains.

Accordingly, the invention provides method for decreasing theimmunogenicity of antibody variable domains comprising a variable lightchain and/or a variable heavy chain, wherein the method comprises thestep of substituting one or more amino acid residues of the variablelight chain and/or the variable heavy chain, said residue being presentat the interface between the variable chain and the constant chain of acorresponding full-length antibody or Fab.

In one aspect, the antibody variable domain is an scFv, an Fv fragmentor a single domain antibody, in particular an scFv.

In one aspect, one or more amino acid residues of the variable lightchain and/or the variable heavy chain to be substituted are consensusresidues of the respective subtype.

In another aspect, the one or more amino acid residues to be substitutedare Leucine (L), Valine (V), Aspartic acid (D), Phenylalanine (F),Arginine (R) and/or Glutamic Acid (E).

In certain aspects, the one or more amino acid residues of the variablelight chain are at positions 99, 101 and/or 148 (AHo numbering). Inother aspects, the one or more amino acid residues of the variable heavychain are at one or more positions 12, 97, 98, 99, 103, and/or 144 (AHonumbering).

In still another aspect, the one or more amino acid residues to besubstituted in the variable heavy chain is (a) Leucine (L) at heavychain amino acid position 12; (b) Valine (V) at heavy chain amino acidposition 103; and/or (c) Leucine (L) at heavy chain amino acid position144.

In another aspect, the invention provides antibody variable domainsobtainable by the method disclosed herein, and pharmaceuticalcompositions comprising said antibody variable domains.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings.

FIG. 1a shows a schematic view of the bridging ELISA used to detectpre-existing anti-scFv antibodies. 1: plate surface, 2: scFv903; 3:Anti-Drug Antibody (ADA); 4: biotinylated scFv903; 5: StreptavidinPoly-HRP (Horse Radish Peroxidase).

FIG. 1b shows the principle for confirmation assessment where ADAbinding to the drug and biotinylated scFv903 was competed with an excessof scFv903, 34 max (791), scFv903_DHP (961) and scFv105 (100 mcg/ml).

FIG. 2 shows signal intensity of 149 individual sera in a colorimetricassay (briding ELISA) to detect anti-scFv 903 antibodies. Signals abovethe assay cut point indicate presence of anti-scFv 903 antibodies (ADAs)in the respective serum sample. Roughly 30% of tested sera samples werepositive in the assay.

FIG. 3 shows a variable light chain sequence alignment of solventexposed positions of four different scFvs. Upper panel: amino acids ineach scFv differing in type from the respective amino acid in scFv 903are given in bold. The lower panel indicates the epitope category towhich each individual position was associated, and the percentage ofhuman sera that showed binding to the respective epitope category.

FIG. 4 shows a variable heavy chain sequence alignment of solventexposed positions of four different scFvs. Upper panel: amino acids ineach scFv differing in type from the respective amino acid in scFv 903are given in bold. The lower panel indicates the epitope category towhich each individual position was associated, and the percentage ofhuman sera that showed binding to the respective epitope category.

FIGS. 5a-5b show the modeled molecular structure of scFv 903. FIG. 5a :front view; FIG. 5 b: 180° view. Gray: residues potentiallyparticipating in the epitope category β; black: residues potentiallyparticipating in the epitope category α.

DISCLOSURE OF THE INVENTION

So that the invention may be more readily understood, certain terms arefirst defined. Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, suitablemethods and materials are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

The expression “immunogenicity” as used herein means the occurrence of Bcell or antibody epitopes on a protein administered to a subject,whereas such B cells or antibodies (also referred to as anti-drugantibodies; ADAs) may have existed prior to the administration of saidprotein.

The extent of such immunogenicity can be determined by an ELISA assayand can be expressed as the percentage of human sera that containmeasurable amounts of pre-existing ADAs. A reduction of immunogenicitybetween a protein and a corresponding protein being engineered with thegoal to reduce its immunogenicity can be measured by comparing thepercentage of serum samples containing ADAs against the engineeredprotein with the percentage of serum samples containing ADAs against theoriginal protein. A lower number or percentage of positive serum samplesfor the engineered protein indicates a reduction of immunogenicity forthe engineered protein. A more sensitive measurement, which can beapplied on the basis of a single serum sample, employees a competitionELISA setup. In such competition ELISA the engineered protein competeswith the original protein for binding of ADAs in the test serum. Thelower the ability of the engineered protein to compete with the originalprotein, the more successful the immunogenicity was reduced.

Preferably, the extent of immunogenicity reduction is referred to aspercentage of serum samples in which the engineered protein is no moreable to effectively compete with the original protein. Effectivecompetition is defined by a threshold (a relative signal from thecompetition ELISA), whereas −100 indicates a perfect competitor (noreduction of immunogenicity) and 0 indicates no competition at all(complete absence of ADA epitopes). Typically, such threshold foreffective competition can be −90, −80, −70, −60, 50, −40, −30, −20, −10or >−10.

“Interface” or “interface-interface” as used herein refers to thoseregions localized between the variable domains and the constant regions1 (CL1 or CH1) of a full length antibody or between the Fab portion andthe Fc domain (CH2 and CH3).

“ADA”, as used herein, is an abbreviation for anti-drug antibodies whichrefers to pre-existing antibodies in the serum or sera of patients.

The term “antibody variable domain” (V-Domain) refers to a molecule thatcontains all or a part of the antigen binding site of an antibody, e.g.,all or part of the heavy and/or light chain variable domain, such thatthe antibody variable domain specifically recognizes a target antigen.The term thus corresponds to the V-J-REGION or V-D-J-REGION of theimmunoglobulin. These V-Domains are designated as: VL (V-Domain of anIg-light chain) or VH (V-Domain of an Ig-heavy chain). Non-limitingexamples of antibody variable domains include

-   -   (i) Fv fragments comprising the V_(L) and V_(H) domains of a        single arm of an antibody,    -   (ii) single chain Fv fragments (scFvs),    -   (iii) single domain antibodies such as Dab fragments (Ward et        al., (1989) Nature 341:544-546), which consist of a VH or a VL        domain, Camelid (see Hamers-Casterman, et al., Nature        363:446-448 (1993), and Dumoulin, et al., Protein Science        11:500-515 (2002)) or Shark antibodies (e.g., shark Ig-NARs        Nano-bodies®).

The term “antibody framework” or “framework” as used herein refers tothe part of the variable domain, either VL or VH, which serves as ascaffold for the antigen binding loops of this variable domain (Kabat,E. A. et al., (1991) Sequences of proteins of immunological interest.NIH Publication 91-3242).

The term “antibody CDR” or “CDR” as used herein refers to thecomplementarity determining regions of the antibody which consist of theantigen binding loops as defined by Kabat E. A. et al., (1991) Sequencesof proteins of immunological interest. NIH Publication 91-3242). Each ofthe two variable domains of an antibody Fv fragment contain, forexample, three CDRs.

The term “single chain antibody” or “scFv” refers to a moleculecomprising an antibody heavy chain variable region (V_(H)) and anantibody light chain variable region (V_(L)) connected by a linker. SuchscFv molecules can have the general structures:NH2-V_(L)-linker-V_(H)-COOH or NH2-V_(H)-linker-V_(L)-COOH.

The term “subtype” refers to a set of V-DOMAINs which belong to the samegroup, in a given species, and which share high percentage of identity.The term “subtype” refers to the subtype defined by the respectiveconsensus sequence as defined in Knappik (2000). The term “subfamily” or“subclass” is used as synonym for “subtype”. The term “subtype” as usedherein refers to sequences sharing the highest degree of identity andsimilarity with the respective consensus sequence representing theirsubtype. To which “subtype” a certain variable domain belongs to isdetermined by alignment of the respective sequence with either all knownhuman germline segments or the defined consensus sequences of therespective subtype and subsequent association to a certain subtype basedon greatest homology. Methods for determining homologies and grouping ofsequences by using search matrices, such as BLOSUM (Henikoff 1992) arewell known to the person skilled in the art.

The “consensus residue” at a given position can be determined bygenerating the amino acid consensus sequence of a given subtype. “Aminoacid consensus sequence” as used herein refers to an amino acid sequencethat can be generated using a matrix of at least two, and preferablymore, aligned amino acid sequences, and allowing for gaps in thealignment, such that it is possible to determine the most frequent aminoacid residue at each position. The consensus sequence is that sequencewhich comprises the amino acids which are most frequently represented ateach position. In the event that two or more amino acids are equallyrepresented at a single position, the consensus sequence includes bothor all of those amino acids. The amino acid sequence of a protein can beanalyzed at various levels. For example, conservation or variability canbe exhibited at the single residue level, multiple residue level,multiple residue with gaps etc. Residues can exhibit conservation of theidentical residue or can be conserved at the class level. Other classesare known to one of skill in the art and may be defined using structuraldeterminations or other data to assess substitutability. In that sense,a substitutable amino acid can refer to any amino acid which can besubstituted and maintain functional conservation at that position. Asused herein, when one amino acid sequence (e.g., a first VH or VLsequence) is aligned with one or more additional amino acid sequences(e.g., one or more VH or VL sequences in a database), an amino acidposition in one sequence (e.g., the first VH or VL sequence) can becompared to a “corresponding position” in the one or more additionalamino acid sequences. As used herein, the “corresponding position”represents the equivalent position in the sequence(s) being comparedwhen the sequences are optimally aligned, i.e., when the sequences arealigned to achieve the highest percent identity or percent similarity.

The AHo numbering scheme used throughout the description is described inA. Honegger and A. Plückthun (2001), J. Mol. Biol. 309: 657-670.

The term “patient” refers to a human or to a non-human animal.

The term “treat”, “treating” or “treatment” refers to therapeutic and/orpreventive measures with the aim to prevent, cure, delay, reduce theseverity of or ameliorate one or more symptoms of the disorder orrecurring disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

“Hydrophilic” amino acids are polar and electrically charged aminoacids, such as Asp, Glu, Lys, Arg and His.

Amino acids that are polar and uncharged are Gly, Ser, Thr, Cys, Asp,Gln and Tyr.

“Hydrophobic” amino acids are typically non polar amino acids such asAla, Val, Leu, Ile, Met, Phe, Trp and Pro.

In a first aspect, a method for decreasing the immunogenicity of anantibody variable domain is disclosed. The antibody variable domaincomprises a variable light chain and/or a variable heavy chain, and themethod comprises the step of substituting one or more amino acidresidues of the variable light chain and/or the variable heavy chain,said residue being present at the interface between the variable chainand the constant chain of a corresponding full-length antibody (or Fab,i.e. any antibody or antibody fragment comprising a constant domain orparts thereof).

Said one or more amino acid residues selected for substitution arepreferably those which are present at the interface between the variablechain and the constant chain of the corresponding full-length antibody(or Fab, i.e. any antibody or antibody fragment comprising a constantdomain or parts thereof) and are solvent exposed in an antibody variabledomain, such as a scFv. Said interface is also termed V/C domaininterface.

The antibody variable domain is e.g. an scFv, an Fv fragment or a singledomain antibody, preferably a scFv.

Of particular interest are the amino acid residues at positions thatform discontinuous, i.e. conformational, B-cell epitopes. Such residuesinclude those found at the following positions (AHo numbering):

variable light chain positions 99, 101 and/or 148; and

variable heavy chain positions 12, 97, 98, 99, 103, and/or 144.

Residue positions 99, 101 and 148 (AHo numbering) of the light chain, aswell as residue positions 12, 98, 103, and 144 (AHo numbering) of theheavy chain: are known from Nieba et al. (1997) Protein Eng., April;10(4):435-44 (also disclosed in U.S. Pat. No. 6,815,540) for improvingfolding behavior of antibodies by protein engineering. Nieba proposes tosubstitute hydrophobic amino acids by hydrophilic ones at the indicatedpositions; however, the document is silent that these substitutions mayhave an influence on the immunogenicity of the molecule. Moreover, theauthors highlight that not all of these hydrophobic residues are equallygood candidates for replacements. While the existence of the hydrophobicpatches is preserved in all antibodies, their exact position and extentvaries.

As known in the art, in particular amino acids which

-   -   (i) are present in a turn region of the secondary structure,    -   (ii) have a large, flexible side chain or a bulky side chain, or    -   (iii) are hydrophobic

are prone to be part of a B-cell epitope and thus elicit an immunogenicreaction. By removing immunogenic amino acids, B-cell epitopes areinterrupted and the patient's tolerance to the antibody variable domaincan be enhanced.

Preferably, the selected one or more amino acid residues are substitutedby an amino acid which is less immunogenic than the selected aminoacids, i.e. does not elicit an immune response or elicits a weak immuneresponse. Such less immunogenic amino acids are those that reduce ADAreactivity compared with ADA reactivity to the antibody variable domaincontaining the original (i.e. un-substituted) amino acid.

Immunogenicity, i.e. the property to induce an antibody response withinthe patient's body, can e.g. be predicted by its antigenicity, i.e. thereactivity with pre-existing antibodies. The antigenicity may e.g. bedetermined by ADA reactivity via a bridging ELISA (see example 1 andFIG. 1), using sera from donors which potentially comprise pre-existingantibodies. Hence, for the evaluation of less immunogenic amino acids,the antibody variable domain may be mutated at the indicated positions.The effect of such mutations on immunogenicity can be assessed bycompeting the signal of the progenitor antibody in the bridging ELISAwith the presumably less immunogenic, engineered derivative thereof, asdescribed herein. Binding of ADAs against an antibody can also beassessed by the use of label-free binding assays, such as surfacePlasmon resonance, fluorescence resonance energy transfer (FRET),calorimetric assays and others.

In one embodiment, amino acids chosen for being substituted are at oneor more positions selected from the group consisting of variable lightchain residues 99, 101 and 148 and variable heavy chain residues 12, 97,98, 99, 103 and 144.

Preferred amino acids chosen for substitutions are surface exposed butwould be hidden by the constant domain in a corresponding full-lengthantibody or Fab.

In one embodiment, the one or more amino acid residues of the variablelight chain and/or the variable heavy chain to be substituted areconsensus residues of the respective subtype. For example, preferredamino acids chosen for substitutions are Leucine (L), Valine (V),Aspartic acid (D), Phenylalanine (F), Arginine (R), and/or Glutamic Acid(E).

More preferably, the one or more amino acid residues chosen for beingsubstituted are selected from the group consisting of variable lightchain residues D99, F101 and L148 and variable heavy chain residues L12,R97, A98, E99, V103 and L144.

Even more preferably, Leucine (L), Valine (V), Phenylalanine (F) and/orAlanine (A) are substituted by polar amino acids, preferably by serine(S) and/or threonine (T).

In particular, the DHP motif as described in PCT/CH2009/00022 has beenunexpectedly found to decrease the immunogenicity of antibody variabledomains without having an adverse effect on the thermal stability, therefolding, the expression yield, the aggregation and/or the bindingactivity of the antibody variable domain. Said DHP motif comprises theamino acid residues of the variable heavy chain 12, 103 and 144 (AHonumbering) at which the following amino acids are present:

(a) Serine (S) at heavy chain amino acid position 12;

(b) Serine (S) or Threonine (T) at heavy chain amino acid position 103;and/or

(c) Serine (S) or Threonine (T) at heavy chain amino acid position 144.

PCT/CH2009/00022 does not provide any hint that the taught modificationsare suitable to decrease the immunogenicity of antibody variabledomains.

The DHP motif is located at the V/C interface of a Fab fragment andbecomes solvent exposed upon removal of the constant domains. Thus in apreferred embodiment of the present invention, one or more amino acidresidues are selected for substitution from the group consisting of thevariable heavy chain 12, 103 and 144 (AHo numbering). Preferably,

(a) Leucine (L) is present at heavy chain amino acid position 12;

(b) Valine (V) is present at heavy chain amino acid position 103; and/or

(c) Leucine (L) is present at heavy chain amino acid position 144.

These residues are highly conserved in human frameworks. Thus,substituting one or more of said residues provides a general solution tode-immunize antibody variable domains without affecting the biophysicalproperties of the molecule, and the method disclosed herein isapplicable to any framework of an antibody variable domain. Preferably,the residues present at the indicated position(s) are substituted by

(a) Serine (S) at heavy chain amino acid position 12;

(b) Serine (S) or Threonine (T) at heavy chain amino acid position 103;and/or

(c) Serine (S) or Threonine (T) at heavy chain amino acid position 144.

Even more preferably, the following substitutions are made: L12S, V103Tand/or L144T.

The antibody variable domain may be directed against any target, andspecifically binds said target. Exemplary examples of targets include,but are not limited to: a transmembrane molecule, a receptor, a ligand,a growth factor, a growth hormone, a clotting factor, an anti-clottingfactor, a plasminogen activator, a serum albumin, a receptor for ahormone or a growth factor, a neurotrophic factor, a nerve growthfactor, a fibroblast growth factor, transforming growth factor (TGF), aCD protein, an interferon, a colony stimulating factor (CSF), aninterleukin (IL), a T-cell receptor, a surface membrane protein, a viralprotein, a tumor associated antigen, an integrin or an interleukin,VEGF; a renin; a human growth hormone; a bovine growth hormone; a growthhormone releasing factor; parathyroid hormone; thyroid stimulatinghormone; a lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulinB-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; clotting factor VIIIC; clotting factorIX; tissue factor (TP); von Willebrands factor; Protein C; atrialnatriuretic factor; a lung surfactant; urokinase; human urine;tissue-type plasminogen activator (t-PA); bombesin; thrombin;hemopoietic growth factor; tumor necrosis factor-alpha or -beta;enkephalinase; RANTES (Regulated on Activation Normally T-cell Expressedand Secreted); human macrophage inflammatory protein (MIP-1)-alpha;human serum albumin; Muellerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA); CTLA-4; inhibin; activin; vascularendothelial growth factor (VEGF); protein A or D; a rheumatoid factor;bone-derived neurotrophic factor (BDNF); neurotrophin-3, -4, 5, or -6(NT-3, NT-4, NT-5, or NT-6); NGF-beta; platelet-derived growth factor(PDGF); aFGF; bFGF; epidermal growth factor (EGF); TGF-alpha; TGF-beta,including TGFbeta1, TGF-beta2, TGF-beta3, TGF-beta14, or TGF-beta5;insulin-like growth factor-I or -II (IGF-I or IGF-II); des(1-3)-IGF-I(brain IGF-I), an insulin-like growth factor binding protein,erythropoietin; an osteoinductive factor; an immunotoxin; a bonemorphogenetic protein (BMP); interferon-alpha, -beta, or -gamma; M-CSF,GM-CSF or G-CSF; IL-1 to IL-10; superoxide dismutase; decay acceleratingfactor; an AIDS envelope protein; a transport protein; a homingreceptor; an addressin; a regulatory protein; CD3, CD4, CD8, CD11a,CD11b, CD11c, CD18, CD19, CD20, CD34, CD40, or CD46, an ICAM, VLA-4 orVCAM; or HER2, HER3 or HER4 receptor; a member of the ErbB receptorfamily; an EGF receptor; HER2, HER3 or HER4 receptor; a cell adhesionmolecule; LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, alpha4/beta7integrin or alphav/beta3 integrin; an alpha or beta subunit of a celladhesion molecule; antibodies); a growth factor, VEGF; tissue factor(TF); TGF-beta; alpha interferon (alpha-IFN); IL-8; IgE; blood groupantigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB)receptor; mpl receptor; CTLA4 or protein C.

In another embodiment, the invention provides an antigen bindingfragment obtainable by the method disclosed herein. Said antigen bindingfragment may e.g. be used for therapeutic or diagnostic applications.

The sequences used in the Examples herein include:

>903 or 578minmax (SEQ ID NO: 1)EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS >791 or 34max (SEQ ID NO: 2)MEIVMTQSPSTLSASLGDRVIITCQSSQSVYGNIWMAWYQQKSGKAPKLLIYQASKLASGVPSRFSGSGSGAEFSLTISSLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFTISRSYWICWVRQAPGKGLEWVACIYGDNDITPLYANWAKGRFPVSTDTSKNTVYLQMNSLRAEDTAVYYCARLGYADYAYDLWGQGTLVTVSS >scFv105 (SEQ ID NO: 3)DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS >961 or 578minmaxDHP (SEQ ID NO: 4)EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTATY YCAGGDHNSGWGLDIWGQGTTVTVSS

Example 1 Anti-Drug-Antibody Bridging ELISA (ADA-ELISA) 1.1 Background

Pre-existing antibodies against a monoclonal antibody may be eitherdirected to constant regions, to variable domain framework positions orto the antigen binding loops, the CDRs. Pre-existing antibodies bindingspecifically to Fv fragments but not to IgGs are likely to recognizeregions that were formerly shielded in the IgG. Such regions are mainlythe domain interfaces localized between the variable domains and theconstant regions 1 (CL1 or CH1) or between the Fab portion and the Fcdomain (CH2 and CH3). Antibodies that recognize such interfaces areformat specific in all likelihood. Since the framework sequence ofscFv903 is highly conserved in humans, it appears likely that thepre-existing antibodies to scFv903 in human sera either bind to CDRs orto V/C-interface residues. The epitopes for such pre-existinganti-scFv903 antibodies were characterized in a sandwich ELISA byassessing the potential of a variety of scFvs to compete with binding ofanti-drug antibodies (ADA) to ESBA903. The scFvs tested were:

scFvs containing the same framework as scFv903 but different CDRs(34_max (791)),

scFvs with a different framework than scFv903 and different CDRs(scFv105), and

an scFv903 variant (scFv903 DHP (961)) containing substitutions in theformer V/C interface.

The ELISA developed for screening of anti-scFv903 antibodies is aquasi-quantitative assay and was developed in a bridging format (seeFIG. 1) which allows detection of responses of all antibody isotypesfrom different species.

Briefly and with reference to FIG. 1, microtitre plates were coated withscFv 903 1, 2 to which samples containing anti-scFv 903 antibodies 3, 6were bound. As a first detection agent, biotinylated scFv 903 4 was usedto detect any bound scFv 903/anti-scFv 903 complexes which in turn weredetected by a second detection agent 4, Streptavidin Poly-HRP 5. Theamount of anti-scFv 903 antibodies present in the quality control andsamples was determined using peroxidase (POD) substrate(3,3′-5,5′Tetramethylbenzidine (TMB)).

The development of the ADA ELISA was performed with a positive controlantibody termed AB903-3. The Anti-scFv 903 antibody stock (rabbitpolyclonal Anti-scFv 903 IgG termed AB903-3) was developed byimmunization of rabbit with scFv 903 and subsequentaffinity-purification of the serum (Squarix Biotechnology). As depictedin FIG. 1b , epitopes of pre-existing antibodies on scFv 903 werecharacterized by competition of ADA binding to scFv 903 with the scFvsdescribe above.

1.2 Assay Procedure

A microtitre plate (Nunc Maxisorp) was coated with 0.1 mcl/ml scFv 903in PBS (Dulbecco, Sigma). The sealed plate was incubated overnight at 4°C.

The plate was washed three times with 300 mcl/well wash buffer (TBST0.005% Tween (20) in an Atlantis Microplate Washer (ASYS). Non-specificsites were blocked with 280 mcl/well blocking buffer (PBS, 10 mg/ml BSA1% (w/v), 0.1 ml/50 ml Tween 20 (0.2%, v/v). The sealed plate wasincubated for 1.5 hours at room temperature (25° C.) with shaking.Subsequently, the plate was again washed three times as indicated above.

The analyte control (either an affinity purified rabbit polyclonalAnti-scFv 903 IgG termed AB903-3 or human sera) was added in threedifferent concentrations:

HiQC: 2500 ng/ml AB903

MeQC: 500 ng/ml AB903

LoQC: 250 ng/ml AB903

QC were spiked in the respective NSB serum pool (pool of all sera usedfor determination of the assay cut point, >30). The samples to bemeasured were applied in a 1 to 10 dilution. 50 mcl of sample wereapplied per well; the sealed plate was incubated 2.0 hours at roomtemperature (25° C.).

As indicated above, the plate was washed three times with washingbuffer. As a first detection agent, biotinylated scFv 903 (500 mcgprotein biotinylated with Lightning-Link kit (protocol: Lightning-Link™Biotin Conjugation Kit, Type A, #704-0015, Innova Biosciences) wasadded. For said purpose, biotinylated scFv 903 was diluted in dilutionbuffer at a concentration of 250 ng/ml (PBS, 10 mg/mL BSA 1% (w/v), 0.1ml/50 ml Tween 20 (0.2% v/v)). 50 mcl/well was added. The sealed platewas incubated 1.0 hour at room temperature (25° C. nominal) undershaking.

The plate was again washed three times as indicated above. The seconddetection agent, Streptavidin-Poly-HRP (Stereospecific DetectionTechnologies, 1 mg/ml) was diluted 1:5′000 in dilution buffer and 50 mclwere added per well. The sealed plate was incubated 1.0 hour at roomtemperature (25° C. nominal) under shaking.

For the detection, the plate was washed three times with 300 mcl/wellwash buffer as indicated above. Subsequently, the plate was washed twicewith 300 mcL/well ddH2O. Then, 50 mcl/well POD (TMB) substrate at roomtemperature was added. After incubation for 3-6 minutes (the maximalincubation time of 30 min should not be exceeded), the reaction wasstopped by adding 50 mcl/well 1M HCL. If the color reaction was veryintense, the reaction was stopped earlier by 50 mcl/well 1M HCL.

Using a microtiter plate reader from Tecan Sunrise, the plate was readat 450 nm. Typically, the reaction was performed in triplicate for eachquality control, NSB and individual serum sample. The readings wereaveraged.

1.3 Assay Cut Point (ACP) Determination

For determination of positives, an assay cut point was establishedduring assay development. The cut point of an assay is the level ofresponse of the assay at or above which a sample is defined to bepositive and below which it is defined to be negative. Using arisk-based approach, it is appropriate to have 5% false positives,rather than any false negatives. This was done with a parametricapproach using the mean absorbance plus 1.645 Standard deviations, where1.645 is the 95th percentile of the normal distribution. All individualswith OD≥3*standard deviation were excluded and a new assay cut point wascalculated. If ODs of maximally 5% of the individuals are above the ACP,the calculated value can be used as ACP. In the contrary, the exclusioncriteria to the remaining individuals were applied again and the processwas repeated until maximally 5% of the individuals had an OD above theACP.

After exclusion of 34 out of 149 individual sera, the assay cut pointwas corrected to 0.110. 40 sera (26.8%) showed an OD higher than theassay cut point (see FIG. 2). LoQC, MeQC and HiQC samples were includedfor monitoring of assay performance. For preparation of the NSB the 34sera removed for statistical evaluation of the assay cut point wereexcluded from the serum pool. The mean NSB result was 0.073 resulting ina normalisation factor of 1.50.

1.4 Determination of Normalisation Factor and Plate Specific Cut Point

Once an ACP was determined with a NSB, the normalisation factor wasapplied to calculate the plate specific cut point for subsequentmeasurements with the same NSB. To determine the normalisation factor,the OD values of a NSB were assessed. Three replicates (triplicates) ofthe NSB were analysed on each plate. The normalisation factor wasdefined as the assay cut point divided by the mean absorbance of NSB.The plate specific cut point for each plate was calculated as follows:

Plate specific cut point=NSB absorbance*normalisation factor.

1.5 Confirmatory Assay

In case of detected ADAs in the serum, a confirmatory assay proves thatthe antibodies found to be positive in the ADA bridging ELISA arespecific to scFv 903. The confirmatory assay was similar to thescreening assay, except that positive samples were mixed andpre-incubated with assay buffer containing scFv 903 or just assay bufferprior to analysis. For this purpose, reference material was spiked indilution buffer or human serum to a concentration of the LoQC, MeQC andHiQC level. These samples were then diluted 1 in 2 with either buffer orbuffer containing 10 mcg/ml, 100 mcg/ml or 1 mcg/ml (for human serum)scFv 903. Samples were incubated at RT for approximately 60 minutes toallow binding of scFv 903 to ADAs present in the sample. Samples werediluted further in buffer prior to loading onto the plate in order thatthe overall matrix dilution was at the minimum. scFv 903 preventsbinding of the ADAs to scFv 903 coated on the plate (see also FIG. 1).Therefore, a change in OD values of >30% between sera diluted 1 in 2with buffer and sera diluted 1 in 2 with buffer containing scFv 903 wasdefined as minimal inhibition to confirm presence of specific anti-scFv903 antibodies. Pre-incubation with scFv 903 at 10 mcg/ml, 100 mcg/ml aswell as 1 mcg/ml resulted in OD changes between 60% and 95% for all 3 QClevels tested. A concentration of 100 mcg/ml was selected for theconfirmation assay.

1.6 Results of the Competition Assay

In order to map the binding sites of anti-scFv 903 antibodies, a set ofdifferent scFvs with known amino-acid sequences as well as the IgGformat of scFv903 was used instead of excess scFv 903 in theconfirmatory assay set up described above. In this experimental setup, agiven test antibody can only compete for binding of scFv 903 toanti-scFv 903 antibodies (ADAs), if the ADAs recognize a similar epitopealso on the test scFv. Thus, a signal reduction in the assay wouldindicate the presence of at least one epitope that is shared betweenscFv 903 and test scFv. The following test antibodies have been used inthis experiment: scFv105, a humanized TNF-inhibitory scFv antibodyfragment containing mouse CDRs grafted onto a human scFv scaffold of thetype Vk1-VH1b; scFv 791, a humanized TNF-inhibitory antibody fragmentcontaining rabbit CDRs grafted on the same scFv scaffold as used forscFv 903 (Vk1-VH3); scFv 961, a derivative of scFv 903 containing threepoint mutations (the DHP-motif) in the region participating in theinterface between variable and constant domain, and scFv903-IgG, the IgGformat of scFv 903.

Correlation of sequence variations between the four tested moleculeswith differences in ADA binding characteristics was used to identify ADAepitopes in the entire scFv scaffold and more specifically in the V-Cinterface. In addition, competition with a full-size version of scFv903(IgG) was used to further confirm the format specificity of thepre-existing ADAs.

A summary of data from competition experiments is shown in table 1.Binding of all but two individual human sera was competed with an excessof scFv903 confirming that these ADAs were specific for ESBA903. Out ofthe 32 human sera specific for scFv 903 only two were not competed byscFv791, indicating that the antibodies present in these human sera (H53and H76) bind to scFv903 CDRs, while all other sera apparently were notCDR specific. About 48% of ADAs did also recognize epitopes on scFv105,although these responses were slightly lower. This suggested that mostof the antibodies are not explicitly framework specific but rather bindto amino acids conserved in different scFv scaffolds. Interestingly, theIgG format of scFv 903 did not significantly compete with scFv 903 forbinding to any of the sera tested. This strongly suggests that themajority of sera bind to the interface between variable and constantregion, which is accessible to ADAs in scFv 903 but not in the IgGformat thereof. Furthermore, scFv 961, differing in only three aminoacids from scFV903 competed binding of only 64% of the ADAs. Theseresponses were generally lower than with scFV903, which implies that thepredominant fraction of pre-existing ADAs bind to to an epitopecomprising these three amino acids that constitute a hydrophobic surfacepatch in the V-C interface of scFv 903. The difference between resultsobtained with scFV105 compared to scFV903 can be explained by thepresence of different patterns of hydrophobic surface patches in thesetwo molecules. In summary, these results show that up to 50% ofpre-existing ADAs in human sera repress sent scFv format specificantibodies.

TABLE 1 Epitope characterization of pre-existing antibodies in humansera. % reduction in OD upon competition with 100 mcg/ml of scFv903, 791(34_max), scFv105 and 961 (scFv903_DHP) and the IgG format of scFv903are given for 34 different human sera with pre-existing anti-scFv903antibodies. human % reduc- % reduc− % reduc− % reduc− sera tion903tion791 tion105 tion961 H3 −79.9 −85.0 −58.8 −1.7 H8 −87.3 −90.7 −23.1−88.6 H14 −96.6 −96.5 −3.6 na H15 −91.1 −90.0 −69.5 na H19 −90.3 −84.3−73.5 −63.7 H20 −91.9 −96.1 −18.8 −23.5 H21 −85.4 −85.3 −3.4 −80.5 H26−92.0 −96.8 −79.9 0.1 H29 −79.4 −78.7 −51.4 −21.9 H40 −95.4 −94.6 3.5−1.7 H46 −79.8 −77.1 −66.7 −40.1 H49 −82.7 −81.2 −26.1 −8.8 H50 −88.7−88.5 −31.1 −72.8 H53 −32.4 −34.6 −18.4 −30.9 H54 −93.1 −91.7 −61.3−69.4 H55 −89.4 −96.0 −71.9 −14.7 H56 −24.4 −15.1 −4.8 −23.8 H59 −97.0−96.4 −93.7 −67.3 H60 −95.6 −94.8 −10.8 −1.2 H63 −96.8 −19.7 −80.5 −35.9H64 −96.9 −95.5 −55.5 −13.7 H66 −97.2 −96.4 −2.6 na H69 −92.2 −74.7−87.6 −6.2 H76 −97.0 −48.6 −65.7 −36.4 H79 −97.1 −96.8 −40.5 −74.1 H80−87.0 −85.3 −50.9 −55.1 H81 −94.7 −94.4 −18.1 −8.8 H86 −86.2 −86.7 Na−67.9 H96 −80.3 −78.5 −29.0 −78.0 H105 −97.2 −96.9 2.4 7.4 H115 −94.7−94.8 −60.4 −31.4 H116 −97.8 −97.2 −43.4 −33.2 H125 −97.9 −97.4 −55.52.7 H135 −92.5 −95.2 −23.1 −74.7

To identify the interaction sites between ADAs and scFv 903, sequencevariations between scFv 903 and the other scFvs (791, 105 and 961) werecorrelated with the specificities of ADAs in the various human sera. Ina first step the sequences of the different scFvs were aligned andsolvent exposed positions were grouped according to sequence differencesbetween scFv 903 and the other scFvs (FIGS. 3 and 4). Herein, “α”represents the group of positions which differ between scFv 961 and allother scFvs, “β” stands for positions at which only scFv105 differs insequence from the other molecules, and “γ” indicates positions at whichscFv 791 is different from all others. Further αβ and αγ describepositions that are conserved in all scFvs except scFv 961 and scFv105 orscFv961 and scFv791, respectively. Similarly, human sera containinganti-scFv903 antibodies were classified by their specificity to theother scFvs as determined in the competition assay, using the sameclassification code as used above for the amino acid positions (seetable 2). In order for a serum to qualify as binding to a given testscFv a minimal signal reduction of 50% in the competition assay was setas threshold. In this study “α” represents human anti-scFv 903 sera thatdid not show binding activity towards scFv 961. “β”-sera did not bind toscFv105 and “γ”-sera did not bind to scFv 791. From the correlation ofsequence analysis and in vitro binding studies it can be concluded thatfor example anti-scFv 903 antibodies in a type “α” human serum interactwith at least one amino acid from the amino acid group “α”. Similarly,sera of any other type interact with at least one amino acid in therespective amino acid group.

The structural analysis and homology model of scFv 903 were done usingDiscover Studio version 2.5.5. The modeled structure was analyzed todetermine which amino acid residues are exposed to solvent, and whichamino acid residues are buried. The calculation was done by determiningthe relative Solvent Accessible Surface (SAS) of each residue withrespect to their maximum possible solvent accessible surface area. Thecutoff was defined as 25%, therefore residues with a relative SAS equalor more than 25% were considered solvent exposed.

TABLE 2 Results of an ELISA in which scFv 903, 791, scFv105 and 961compete with scFv903 for binding to the antibodies in 34 serum samples.Human Antigenic serum scFv903 scFv791 scFv105 scFv961 region H125 −98−97 −55 3 α H116 −98 −97 −43 −33 αβ H105 −97 −97 2 7 αβ H79 −97 −97 −41−74 β H26 −92 −97 −80 0 α H14 −97 −96 −4 nd nd H66 −97 −96 −3 nd nd H59−97 −96 −94 −67 All H20 −92 −96 −19 −23 αβ H55 −89 −96 −72 −15 α H64 −97−96 −55 −14 α H135 −93 −95 −23 −75 β H60 −96 −95 −11 −1 αβ H115 −95 −95−60 −31 α H40 −95 −95 4 −2 αβ H81 −95 −94 −18 −9 αβ H54 −93 −92 −61 −69All H8 −87 −91 −23 −89 β H15 −91 −90 −69 nd nd H50 −89 −89 −31 −73 β H86−86 −87 nd −68 nd H80 −87 −85 −51 −55 All H21 −85 −85 −3 −81 β H3 −80−85 −59 −2 α H19 −90 −84 −73 −64 All H49 −83 −81 −26 −9 αβ H29 −79 −79−51 −22 α H96 −80 −79 −29 −78 β H46 −80 −77 −67 −40 α H69 −92 −75 −88 −6α H76 −97 −96 −18 −36 αγ H53 −32 −35 −18 −31 none H63 −97 −20 −80 −36 αγH56 −24 −15 −5 −24 none

94% of the sera had antibodies that bound specifically to scFv 903 or toscFv 791, showing that most pre-existing antibodies did not bind to CDRsregions. Half (50%) of the human sera did not show or had lessantibodies that bind to scFv 961. Sequence analysis revealed that scFv961 and scFv 903 differ only at positions 12, 103 and 144 in thevariable heavy chain. Thus L12, V103 and L144 in the variable heavychain of scFv 903 are involved in ADA binding (table 2 and FIGS. 4 and5). This finding was further confirmed by the fact that the IgG formatof scFv 903, in which the respective interface residues are not solventaccessible due to the contact with the adjacent constant region, did notcompete with binding of anti-scFv 903 sera to scFv 903.

12% of tested human sera had antibodies against all antigenic regions(α,β,γ).

64% of the sera did not contain or had significantly less antibodiesthat bind to scFv 961 (α). As described above, this scFv differs fromscFv 903 only in 3 residue positions (L125, V103T, L103T) located at theformer variable-constant domain interface. Since these residue positionsat the V/C interface are highly conserved, mutating these positionsalready presents a general solution to de-immunize scFvs withoutaffecting the biophysical properties.

51% of the sera did not contain or had less antibodies against scFv105(β) a scFv of the Vk1-VH1b subtype (different framework). Possibleantigenic regions were identified by sequence alignment (FIGS. 3, 4 and5).

23% of the sera were specific to scFv 903 did, however, neither bind toscFv105 nor to scFv 961 (αβ). There is only one residue position thatdiffers in both scFvs when compared to scFv903. Thus the respectiveamino acid in the variable heavy domain of scF 903 (L12) plays a crucialrole in the interaction between ADAs and scFv 903.

In summary it can be concluded that a) roughly 50% of ADAs bind to anepitope in the interface between variable region and constant region(α), comprising residues at positions 12, 103 and 144 in the variableheavy domain, and b) that mutating these three highly conserved residuessignificantly lowers binding strength and frequency of pre-existingantibodies to scFvs in general. This generic applicability of theDHP-motif to reduce immunogenicity of scFvs is further supported by thehigh frequency of Leucine at position 12, Valine at position 103 andLeucine at position 144 in variable heavy chains of human origin. Asbinding of anti-scFv 903 sera to scFv105 was generally weaker whencompared to scFv 903, substitutions of any solvent exposed residue inscFv 903 towards the respective amino acid in scFv105 could potentiallyeliminate or weaken a B-cell epitope. Of particular interest are residuenumbers 101 and 148 in the variable light chain, as these bulky residuesparticipate in the former constant-variable domain interface (table 4).

TABLE 3 Summary table of tested human sera having antibodies againstdifferent antigenic regions. Category (antigenic region) % of sera All12% α 35% β 51% αβ 23% Nd 12% None  4% αγ  4%

TABLE 4 Frequency of amino acids in human variable domains at selectedpositions. Vk1 VH 101 148 12 103 144 A 0.2 0.1 0.6 C 0.2 D 0.1 E 0.1 0.5F 70.4 1.5 0.4 G 0.1 0.3 H 0.1 I 4.0 0.3 4.3 K 92.3 0.1 0.6 L 1.4 0.256.4 4.6 65.5 M 0.1 0.2 0.6 12.3 10.2 N 1.5 P 0.1 0.6 Q 0.3 R 3.7 0.9 S1.8 0.2 0.1 T 0.1 0.6 4.3 19.2 V 21.5 41.2 73.5 0.6 W 1.5 Y 0.1 0.1

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpracticed within the scope of the following claims.

1. An antibody variable domain comprising a variable light chain havinga substitution at position 99, 101 and/or 148 (Aho numbering), and/or avariable heavy chain comprising substitutions at positions 12, 103, and144 (AHo numbering), the antibody having reduced anti-drug antibody(ADA) reactivity compared with the ADA reactivity of the antibodycontaining the original amino acid at the substituted positions.
 2. Theantibody variable domain of claim 1, being an scFv, an Fv fragment or asingle domain antibody.
 3. The antibody variable domain of claim 1,being an scFv.
 4. The antibody variable domain of claim 1, whereinsubstitution of the variable light chain is at position 101 (AHonumbering).
 5. The antibody variable domain of claim 4, whereinsubstitution of the variable light chain at position 101 is a Serine(S).
 6. A pharmaceutical composition comprising the antibody variabledomain of claim
 1. 7. An antibody variable domain comprising a variablelight chain having a substitution at position 99, 101 and/or 148 (Ahonumbering), and a variable heavy chain comprising Serine (S) at heavychain amino acid position 12, Threonine (T) at heavy chain amino acidposition 103, and Serine (S) at heavy chain amino acid position 144 (AHonumbering).
 8. The antibody variable domain of claim 7, being an scFv,an Fv fragment or a single domain antibody.
 9. The antibody variabledomain of claim 8, being an scFv.
 10. The antibody variable domain ofclaim 7, wherein substitution of the variable light chain is at position101 (AHo numbering).
 11. The antibody variable domain of claim 10,wherein substitution of the variable light chain at position 101 is aSerine (S).
 12. A pharmaceutical composition comprising the antibodyvariable domain of claim
 1. 13. A method of reducing anti-drug antibody(ADA) reactivity of an antibody variable domain comprising a variablelight chain and a variable heavy chain, the method comprising making asubstitution at position 99, 101 or 148 (Aho numbering) of the variablelight chain, making a substitution of Serine (S) at heavy chain aminoacid position 12, Serine (S) at heavy chain amino acid position 103, andSerine (S) at heavy chain amino acid position 144 (AHo numbering). 14.The antibody variable domain of claim 13, being an scFv, an Fv fragmentor a single domain antibody.
 15. The antibody variable domain of claim14, being an scFv.
 16. The antibody variable domain of claim 13, whereinsubstitution of the variable light chain is at position 101 (AHonumbering).
 17. The antibody variable domain of claim 16, whereinsubstitution of the variable light chain at position 101 is a Serine(S).
 18. A pharmaceutical composition comprising the antibody variabledomain of claim 13.